Treatment of skin disorders

ABSTRACT

An effective treatment for skin disorders characterized by abnormal skin cell behavior, including a pharmaceutically effective amount of Halofuginone. Skin disorders which can be treated include keloids, hypertrophic scars, psoriasis, acne, seborrhea and alopecia. Halofuginone can reduce or eliminate clinical symptoms of these disorders, as well as substantially prevent the formation of keloids and hypertrophic scars.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a method and a composition for thetreatment of skin disorders and, more particularly, to a method and acomposition for the treatment and prevention of psoriasis, hypertrophicscars and keloids.

Keloids are benign fibrotic tumors which are believed to arise from thereticular dermis. They are characterized by increased tissue fibrosisand collagen deposition [Friedman, D. W. et al., J. Surg. Res., Vol. 55,p. 214-222, 1993]. Keloids usually first appear when a patient isbetween the ages of 10 and 30 years, and are often associated withtrauma. They occur most commonly on the upper back, anterior chest,shoulders and ear lobes. Keloids are especially frequently seen inpatients of African or Asian descent.

Hypertrophic scars are somewhat related to keloids, in that they arealso characterized by increased tissue fibrosis and collagen deposition[Friedman, D. W. et al., J. Surg. Res., Vol. 55, p. 214-222,1993].Furthermore, hypertrophic scars are also most often seen in patients ofAfrican and Asian descent [Rockwell, W. B. et al., Plastic and Recon.Surg., Vol. 84, p 827-835, 1989]. Although there are certain differencesbetween hypertrophic scars and keloids, such as a lower fibroblastdensity in keloids than in hypertrophic scars, a common mechanism isbelieved to underlie both conditions. Specifically, agenetically-determined aberration of the metabolism ofmelanocyte-stimulating hormone (MSH) is believed to be responsible forboth hypertrophic scars and keloids [Rockwell, W. B. et al., Plastic andRecon. Surg., Vol. 84, p. 827-835, 1989]. Thus, both hypertrophic scarsand keloids represent the effects of genetically abnormal behavior ofskin cells.

Keloids and hypertrophic scars are characterized histologically by arich vasculature, a high mesenchymal cell density, a thickened epidermiscell layer, and an abundance of collagen fibers. In hypertrophic scars,these fibers are loosely arrayed in a swirl-like pattern within bundles.In keloids, these fibers show even less organization, without anydiscrete bundles. By contrast, in normal skin these collagen fibers arearranged in distinct, clearly demarcated bundles.

The formation of both keloids and hypertrophic scars is marked by aninitial infiltration of the traumatized tissue by fibroblasts, which isfollowed by the formation of a dense collagenous meshwork. Collagenproduction, as measured by prolyl hydroxylase activity, was found to beelevated in keloids, as compared to normal skin and normally healingwounds [Cohen, K. I. et al., Surg. Forum, Vol 22, p. 488, 1971].Collagen synthesis was also found to be elevated in hypertrophic scars,but not to as great an extent [Rockwell, W. B. et al., Plastic andRecon. Surg., Vol. 84, p. 827-835, 1989]. The ratio of type I collagento type III collagen was found to be significantly elevated in keloidsbut not hypertrophic scars, due to a specific increase in aα1(I)collagen gene expression, although type III collagen gene expression isalso increased [Friedman, D. W. et al., J. Surg. Res., Vol. 55, p.214-222, 1993; Rockwell, W. B. et al., Plastic and Recon. Surg., Vol.84, p. 827-835, 1989]. Thus, clearly the deposition of collagen plays animportant role in keloid and hypertrophic scar formation.

Similarly, psoriasis is also characterized by genetically-determinedabnormal behavior of skin cells. Psoriasis is clinically marked byextensive scaling and a thickened epidermis [G. D. Weinstein and J. L.McCullough, Cell Proliferation Kinetics, p. 327-342]. These clinicalmanifestations are caused by hyperproliferation of epidermal cells. Thishyperproliferation is also seen in non-psoriatic skin of psoriaticpatients, indicating that the genetic defect is also present inapparently “normal” skin cells of psoriatic patients [G. D. Weinsteinand J. L. McCullough, Cell Proliferation Kinetics, p. 327-342]. Althoughcollagen also plays a role in the etiology of psoriasis, the abnormalhyperproliferation of epidermal cells is linked to the increaseddeposition of a number of extracellular matrix components, includingcollagen. Thus, clearly the inhibition of these extracellular matrixcomponents could be an important factor in the inhibition ofhyperproliferation by genetically abnormal psoriatic cells.

Keloids, hypertrophic scars and psoriasis thus have a number ofcharacteristics in common. First, they represent a significant cosmeticproblem, particularly on the face where they can be highly disfiguringand a source of considerable distress to the patient. Second, they canalso be a source of discomfort through pruritus and even pain. Indeed,both keloids and hypertrophic scars can become so large that they arecrippling [D. D. Datubo-Brown, Brit. J. Plas. Surg., Vol 43, p. 70-77,1990]. Furthermore, although keloids on the cornea are rare, they canpotentially result in blindness [D. D. Datubo-Brown, Brit. J. Plas.Surg., Vol 43, p. 70-77, 1990). Third, collagen plays a crucial, ifvaried, role in the development of all three conditions. Finally, allthree conditions are caused by a genetic defect in skin cells, whichcauses these cells to show abnormal behaviors.

Unfortunately, currently available treatments to inhibit the formationand growth of keloids and hypertrophic scars, and to treat psoriasis,are not completely successful. For example, surgery can be used toreduce the size or extent of the lesion, while physical pressure can beused to reduce the size and extent of keloids and hypertrophic scars, aswell as to prevent their initial formation [D. D. Datubo-Brown, Brit. J.Plas. Surg., Vol 43, p. 70-77, 1990). However, neither treatment canprevent the lesion from recurring, and surgery especially carries a riskof increased morbidity.

Other forms of treatment include the administration of corticosteroids.For example, triamcinolone appears to reduce the size of keloids andhypertrophic scars by increasing the rate of collagen degradation[Rockwell, W. B. et al., Plastic and Recon. Surg., Vol. 84, p. 827-835,1989]. However, the side effects of such medications are potentiallydangerous and are not universally successful. Other treatments, such asradiation, also showed variable effectiveness and are associated withother potential side effects [Rockwell, W. B. et al., Plastic and Recon.Surg., Vol. 84, p. 827-835, 1989]. Thus, clearly improved treatments forkeloids and hypertrophic scars are required.

As noted above, collagen synthesis and deposition plays an importantrole in keloid and hypertrophic scar formation, as well as in the cellhyperproliferation associated with psoriasis. The synthesis of collagenis also involved in a number of other pathological conditions,particularly those associated with primary or secondary fibrosis. Thecrucial role of collagen in fibrosis has prompted attempts to developdrugs that inhibit its accumulation [K. I. Kivirikko, Annals ofMedicine, Vol. 25, pp. 113-126 (1993)].

Such drugs can act by modulating the synthesis of the procollagenpolypeptide chains, or by inhibiting specific post-translational events,which will lead either to reduced formation of extra-cellular collagenfibers or to an accumulation of fibers with altered properties.Unfortunately, only a few inhibitors of collagen synthesis areavailable, despite the importance of this protein in sustaining tissueintegrity and its involvement in various disorders.

For example, cytotoxic drugs have been used in an attempt to slow theproliferation of collagen-producing fibroblasts (J. A. Casas, et al.,Ann. Rhern. Dis., Vol. 46, p. 763 (1987)], such as colchicine, whichslows collagen secretion into the extracellular matrix [D. Kershenobich,et aI., N. Engl. J. Med., Vol. 318, p. 1709 (1988)], as well asinhibitors of key collagen metabolism enzymes [K. Karvonen, et al.,J.Biol Chem., Vol. 265, p. 8414 (1990); C. J. Cunliffe, et al., J. Med.Chem., Vol. 35, p.2652 (1992)].

Unfortunately, none of these inhibitors are collagen-type specific.Also, there are serious concerns about the toxic consequences ofinterfering with biosynthesis of other vital collagenous molecules, suchas Clq in the classical complement pathway, acetylcholine esterase ofthe neuro-muscular junction endplate, conglutinin and pulmonarysurfactant apoprotein.

Other drugs which can inhibit collagen synthesis, such as nifedipine andphenytoin, inhibit synthesis of other proteins as well, therebynon-specifically blocking the collagen biosynthetic pathway [T. Salo, etal., J. Oral Pathol. Med., Vol. 19, p. 404 (1990)].

Collagen cross-linking inhibitors, such as β-aminopropionitrile, arealso non-specific, although they can serve as useful anti-fibroticagents. Their prolonged use causes lathritic syndrome and interfereswith elastogenesis, since elastin, another fibrous connective tissueprotein, is also cross-linking. In addition, the collagen cross-linkinginhibitory effect is secondary, and collagen overproduction has toprecede its degradation by collagenase. Thus, a type-specific inhibitorof the synthesis of collagen itself is clearly required as ananti-fibrotic agent.

Such a type-specific collagen synthesis inhibitor is disclosed in U.S.Pat. No. 5,449,678 for the treatment of fibrotic conditions. Thisspecific inhibitor is a composition with a pharmaceutically effectiveamount of a pharmaceutically active compound of a formula:

wherein:

-   -   n is 1 or 2;    -   R₁ is a member of the group consisting of hydrogen, halogen,        nitro, benzo, lower alkyl, phenyl and lower alkoxy;    -   R₂ is a member of the group consisting of hydroxy, acetoxy and        lower alkoxy, and    -   R₃ is a member of the group consisting of hydrogen and lower        alkenoxy-carbonyl.        Of this group of compounds, Halofuginone has been found to be        particularly effective for such treatment.

U.S. Pat. No. 5,449,678 discloses that these compounds are effective inthe treatment of fibrotic conditions such as scleroderma and GraftVersus Host Disease. WO Patent No. 96/06616 further discloses that thesecompounds are effective treatments for restenosis by preventing theproliferation of vascular smooth muscle cells. The two former conditionsare associated with excessive collagen deposition, which can beinhibited by Halofuginone. Restenosis is characterized by smooth musclecell proliferation and extracellular matrix accumulation within thelumen of affected blood vessels in response to a vascular injury [Choiet al., Arch. Surg., Vol. 130, p. 257-261 (1995)]. One hallmark of suchsmooth muscle cell proliferation is a phenotypic alteration, from thenormal contractile phenotype to a synthetic one. Type I collagen hasbeen shown to support such a phenotypic alteration, which can be blockedby Halofuginone [Choi et al., Arch. Surg., Vol. 130, p. 257-261 (1995),WO Patent No. 96/06616]. Thus, Halofuginone can prevent suchredifferentiation of smooth muscle cells after vascular injury byblocking the synthesis of type 1 collagen.

There is thus a widely recognized unmet medical need for an inhibitor ofkeloid and hypertrophic scar formation, as well as for a treatment ofalready formed keloids and hypertrophic scars, which have specificinhibitory effects.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a composition fortreating a skin disorder characterized by substantially abnormal cellbehavior, including a pharmaceutically effective amount of a compound incombination with a pharmaceutically acceptable carrier, the compoundbeing a member of a group having a formula:

wherein:

-   -   n is 1 or 2;    -   R₁ is a member of the group consisting of hydrogen, halogen,        nitro, benzo, lower alkyl, phenyl, and lower alkoxy;    -   R₂ is a member of the group consisting of hydroxy, acetoxy, and        lower alkoxy, and    -   R₃ is a member of the group consisting of hydrogen and lower        alkenoxy alkenoxy-carbonyl.

According to another embodiment of the present invention, there isprovided a composition for substantially preventing a skin disordercharacterized by substantially abnormal cell behavior, including apharmaceutically effective amount of a compound in combination with apharmaceutically acceptable carrier, the compound being a member of agroup having a formula:

wherein:

-   -   n is 1 or 2;    -   R₁ is a member of the group consisting of hydrogen, halogen,        nitro, benzo, lower alkyl, phenyl, and lower alkoxy;    -   R₂ is a member of the group consisting of hydroxy, acetoxy, and        lower alkoxy and    -   R₃ is a member of the group consisting of hydrogen and lower        alkenoxy alkenoxy-carbonyl.

According to yet another embodiment of the present invention, there isprovided a method of manufacturing a medicament for treating a skindisorder characterized by substantially abnormal cell behavior,including the step of placing a pharmaceutically effective amount of acompound in a pharmaceutically acceptable carrier, the compound being amember of a group having a formula:

wherein:

-   -   n is 1 or 2;    -   R₁ is a member of the group consisting of hydrogen, halogen,        nitro, benzo, lower alkyl, phenyl, and lower alkoxy;    -   R₂ is a member of the group consisting of hydroxy, acetoxy, and        lower alkoxy, and    -   R₃ is a member of the group consisting of hydrogen and lower        alkenoxy-carbonyl.

According to still another embodiment of the present invention, there isprovided a method of manufacturing a medicament for substantiallypreventing a skin disorder characterized by substantially abnormal cellbehavior, including the step of placing a pharmaceutically effectiveamount of a compound in a pharmaceutically acceptable carrier, thecompound being a member of a group having a formula:

wherein:

-   -   n is 1 or 2;    -   R₁ is a member of the group consisting of hydrogen, halogen,        nitro, benzo, lower alkyl, phenyl, and lower alkoxy;    -   R₂ is a member of the group consisting of hydroxy, acetoxy, and        lower alkoxy, and    -   R₃ is a member of the group consisting of hydrogen and lower        alkenoxy-carbonyl.

According to another embodiment of the present invention, there isprovided a method for the treatment of a skin disorder characterized bysubstantially abnormal cell behavior in a subject, including the step ofadministering to the subject a pharmaceutically effective amount of acompound having a formula:

wherein:

-   -   n is 1 or 2;    -   R₁ is a member of the group consisting of hydrogen, halogen,        nitro, benzo, lower alkyl, phenyl and lower alkoxy;    -   R₂ is a member of the group consisting of hydroxy, acetoxy and        lower alkoxy, and    -   R₃ is a member of the group consisting of hydrogen and lower        alkenoxy-carbonyl.

In each of the embodiments above, preferably the compound isHalofuginone. Further preferably, the skin disorder is selected from thegroup consisting of psoriasis, keloid, hypertrophic scar, acne,seborrhea and alopecia. Most preferably, the skin disorder is selectedfrom the group consisting of psoriasis, keloid and hypertrophic scar.Hereinafter, the term “keloid-like growth” includes keloid andhypertrophic scar.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

FIGS. 1A and 1B show collagen synthesis in keloid-derived tissue;

FIG. 2 illustrates the inhibitory effect of Halofuginone on collagensynthesis in keloid-derived tissue;

FIG. 3 shows Halofuginone inhibition of sulfate incorporation into ECMof cultured endothelial cells;

FIGS. 4A-4D show inhibition of incorporation of sulfate, proline, lysineand glycine into ECM of bovine corneal endothelial cells byHalofuginone; and

FIGS. 5A-5D show the inhibition of sulfate and glycine incorporationinto rat mesengial cell ECM by Halofuginone.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unexpectedly, Halofuginone has been found to be an effective inhibitorof collagen synthesis by keloid-derived cells. Such an effect was notpredicted by the prior art for the following reasons. First, the priorart did not teach the treatment of keloids or hypertrophic scars withHalofuginone. Second, keloids and hypertrophic scars arise fromgenetically abnormal skin cells. Thus, the behavior of these cells inresponse to Halofuginone cannot be predicted from the response of normalskin cells to Halofuginone. Third, keloids and hypertrophic scars areboth characterized by the abnormal organization of collagen, as well asby oversynthesis of collagen. The prior art did not teach thatHalofuginone would have any effect on pathological processescharacterized by dysfunctional collagen fibril organization. Thus, thefinding that Halofuginone is an effect inhibitor of keloid-relatedpathological processes is both novel and non-obvious.

Such a finding has implications for the treatment of other skinconditions as well, particularly psoriasis. As noted above, psoriasis ischaracterized by hyperplasia of the skin which is enabled by thedeposition of excess extracellular matrix components (ECM), includingcollagen. Furthermore, psoriasis is also caused by genetically abnormalcells. Thus, the use of Halofuginone to treat psoriasis is novel andnon-obvious for the following reasons. First, the prior art did notteach the treatment of psoriasis with Halofuginone. Second, theinhibitory effect of Halofuginone on ECM formation was also notsuggested nor was it taught by the prior art, yet as detailed in theexamples below, Halofuginone completely inhibits deposition of ECMcomponents. Third, as noted above, Halofuginone can alter the behaviorof genetically abnormal skin cells, an effect which was not taught bythe prior art.

Other examples of skin disorders which could be amenable to treatmentwith Halofuginone include acne, seborrhea and alopecia. These conditionsall reflect abnormal skin cell environments. For example, acne is adisorder characterized by excess oil production by the skin, leading tobacterial infection and scar formation if untreated. However, the excessoil production is promoted by the influence of hormones on skin cells,which is one reason adolescents tend to be most affected. Such hormonescause an abnormal environment for the skin cell, so that it in turnbehaves abnormally. As noted above, Halofuginone has been shown to beeffective in the control and inhibition of abnormal skin cell behaviors.Thus, Halofuginone is also a treatment for disorders, such as acne,characterized by such abnormal skin cell behavior.

Halofuginone can therefore be used to both prevent the clinicalmanifestations of skin disorders such as keloids, hypertrophic scars,psoriasis and acne, and to alleviate these disorders once they havearisen. For example, as detailed below, Halofuginone has been shown tobe effective as a pretreatment, before surgery, for anothersurgically-induced pathological process, the formation of adhesions.Thus, Halofuginone is effective as a pretreatment before the appearanceof clinical symptoms, as well as being able to alleviate orsubstantially eliminate such symptoms after they appear.

The present invention may be more readily understood with reference tothe following illustrative examples and figures. It should be noted thatalthough reference is made exclusively to Halofuginone, it is believedthat the other quinazolinone derivatives described and claimed in U.S.Pat. No. 3,320,124, the teachings of which are incorporated herein byreference, have similar properties.

EXAMPLE 1 Collagen Synthesis in Keloid-Derived Tissue

The presence of large amounts of collagen protein, as well as of theexpression of the collagen α1(I) gene, were demonstrated inkeloid-derived tissue. The results are shown in FIGS. 1A and 1B. Theexperiment was conducted as follows.

A keloid, which had arisen in response to the piercing of the ear forinsertion of an earring, was removed from the ear lobe of a 21 year-oldmale. The keloid tissue was sectioned so that histological studies couldbe performed. Briefly, the tissue samples were collected intophosphate-buffered saline (PBS) and fixed overnight in 4%paraformaldehyde in PBS at 4° C. Serial 5 um sections were preparedafter the samples had been dehydrated in graded ethanol solutions,cleared in chloroform and embedded in Paraplast. Differential stainingof collagenous and non-collagenous proteins was performed with 0.1%Sirius red and 0.1% fast green as a counter-stain in picric acid. Thisprocedure stains collagen red [Gascon-Barre, M., et al., J. Histochem.Cytochem., Vol 37, p. 377-381, 1989].

For hybridization with the genetic probe, the sections weredeparafinized in xylene, rehydrated through a graded series of ethanolsolutions, rinsed in distilled water for 5 minutes and then incubated in2× SSC at 70° C. for 30 minutes. The sections were then rinsed indistilled water and treated with pronase, 0.125 mg/ml in 50 mM Tris-HCl,5 mM EDTA, pH 7.5, for 10 minutes. After digestion, the slides wererinsed with distilled water, post-fixed in 10% formalin in PBS andblocked in 0.2% glycine. After blocking, the slides were rinsed indistilled water, rapidly dehydrated through graded ethanol solutions andair-dried for several hours. Before hybridization, the 1600 bp ratcollagen α1(I) insert was cut out from the original plasmid, pUC18, andinserted into the pSafyre plasmid. The sections were then hybridizedwith this probe after digoxigenin-labelling. Alkaline phosphataseactivity was detected in the sections as previously described [Knopov,V., et al., Bone, Vol 16, p. 329S--334S, 1995].

FIG. 1A shows a section of tissue taken from the keloid and stained withSirius red. Most of the keloid tissue was stained with Sirius red exceptthe epidermis, indicating the presence of high concentrations ofcollagen within the tissue. FIG. 1B shows the presence of cells,probably fibroblasts, which express high levels of the collagen α1(I)gene. Thus, clearly the keloid tissue was actively synthesizing collagenand expressing the type I collagen gene.

EXAMPLE 2 Inhibitory Effect of Halofuginone on Collagen Synthesis inKeloid-Derived Tissue

Halofuginone was shown to specifically inhibit collagen synthesis inkeloid-derived tissue. The results are shown in FIG. 2. The experimentwas conducted as follows.

The keloid was removed as described in Example 1 above. Thekeloid-derived cells were incubated with and without Halofuginone for 24hours in 0.5 ml glutamine-free DMEM containing 5% FCS (Fetal CalfSerum), ascorbic acid (50 μg/ml), B-aminopropionitrile (50 μg/ml) and 2μCi of [³H]proline. At the end of incubation, the medium was decantedand the cells were incubated with or without collagenase for 18 hours,followed by precipitation of proteins by the addition of TCA(trichloroacetic acid). The amount of radiolabelled collagen wasestimated as the difference between total labelled-proline containingproteins and those left after collagenase digestion [Granot, I. et al.,Mol. Cell Endocrinol., Vol. 80, p. 1-9, 1991].

The ratio of collagenase digestible to non-collagenase digestibleproteins was found to be higher in the keloid-derived cells than theusual values for normal skin cells. However, FIG. 2 shows thatHalofuginone inhibited the production of collagen, but not ofnon-collagenase digestible proteins. Thus, Halofuginone specificallyinhibited collagen synthesis in keloid-derived cells, and not totalprotein synthesis.

EXAMPLE 3 Halofuginone Inhibition of Sulfate Incorporation into ECM ofCultured Endothelial Cells

As noted above, skin cell hyperproliferation is enabled by thedeposition of ECM components. The following examples illustrate theability of Halofuginone to inhibit such deposition of ECM components,further supporting the use of Halofuginone as a treatment for psoriasis.These examples illustrate the unexpected finding that Halofuginonecompletely abolishes deposition of all ECM components, and not justcollagen, thereby preventing cell proliferation which is enabled by theformation of ECM. Cultures of bovine corneal endothelial cells wereestablished from steer eyes and maintained as previously described [D.Gospodarowicz, et al., Exp. Eye Res., No. 25, pp. 75-99 (1977)]. Cellswere cultured at 37° C. in 10% CO₂ humidified incubators and theexperiments were performed with early (3-8) cell passages.

For preparation of sulfate-labelled ECM (extra-cellular matrix), cornealendothelial cells were seeded into Swell plates at a confluent densityforming, within 4-6 h, a contact inhibited cell monolayer composed ofclosely apposed, and growth arrested cells. Under these conditions, thecells remained viable and retained their normal monolayer configurationand morphological appearance up to a concentration of 2 μg/mlhalofuginone. Na₂[³⁵S]O₄ (540-590 mCi/mmol) was added (40 μCi/ml) oneand five days after seeding and the cultures were incubated withoutmedium change. At various intervals after seeding, the subendothelialECM was exposed by dissolving (5 min., room temperature) the cell layerwith PBS containing 0.5% Triton X-100 and 20 mM NH4OH, followed by fourwashes in PBS [J Vlodavsky, et al., Cancer Res., Vol. 43, pp 2704-2711(1983); I. Vlodavsky, et al., Proc. Natl. Acad. Sci. USA, Vol. 84 pp.2292-2296 (1987)). To determine the total amount of sulfate labeledmaterial, the ECM was digested with trypsin (25 μg/ml 24 h, 37° C.) andthe solubilized material counted in a β-counter.

FIG. 3 shows the almost complete inhibition of sulfate incorporation by1 μg/ml Halofuginone, while 50% inhibition was obtained in the presenceof 0.2 μg/ml of the drug.

EXAMPLE 4 Inhibition of Incorporation of Sulfate, Proline, Lysine andGlycine into ECM of Bovine Corneal Endothelial Cells

Corneal endothelial cells were seeded at a confluent density and grownas described in Example 3 above. The cells were cultured with or withoutHalofuginone in the presence of either Na₂ ³⁵SO₄ (FIG. 4A), ³H-proline(FIG. 4B), ¹⁴C-lysine (FIG. 4C) or ¹⁴C-glycine (FIG. 4D). Eight daysafter seeding, the cell layer was dissolved substantially as describedin Example 3 above. The underlying ECM was then either trypsinized todetermine the effect of Halofuginone on incorporation of labeledmaterial into total protein, substantially as described in Example 3above, or subjected to sequential digestions with collagenase andtrypsin to evaluate the effect of Halofuginone on bothcollagenase-digestible proteins (CDP) and non-collagenase digestibleproteins (NCDP).

As FIGS. 4A-4D show, Halofuginone inhibited the incorporation ofsulfate, proline, lysine and glycine into both CDP and NCDP, reflectinga profound inhibition of matrix deposition. The inhibitory effect ofHalofuginone on deposition of ECM components other than collagen is mostlikely due to the involvement of collagen in the assembly of otherconstituents into the supramolecular structure of the ECM.Alternatively, Halofuginone may affect the synthesis of ECM componentsother than collagen, possibly through a common transcription factor orcytokine such as TGFβ, which affects the synthesis and deposition ofseveral ECM components.

EXAMPLE 5 Inhibition of Sulfate and Glycine Incorporation into RatMesengial Cell ECM

Rat mesengial cells were grown to confluency, 24 hours after seeding.The cells were then cultured with or without Halofuginone in thepresence of either Na₂ ³⁵SO₄ (FIGS. 5A and 5B) or ¹⁴C-glycine (FIGS. 5Cand 5D). Eight days after seeding, the cell layer was dissolved toexpose the underlying ECM, washed and digested with collagenase todetermine the effect of Halofuginone on CDP proteins, as shown in FIGS.5A and 5C. The remaining material was digested with trypsin andsubjected to β-scintillation counting to determine the effect ofHalofuginone on NCDP proteins, as shown in FIGS 5B and 5D.

About 30% inhibition of sulfate incorporation was seen for CDP proteins,while about 70% inhibition was seen for NCDP proteins in the presence of200 ng/ml Halofuginone. It should be noted that the inhibition of ECMdeposition by Halofuginone was not due to its anti-proliferativeactivity since the drug was added to highly confluent, non-dividingcells. Since inorganic sulfate is incorporated primarily into sulfatedglycosaminoglycans and not into collagen, it is conceivable that byinhibiting type I collagen synthesis, Halofuginone interferes with theassembly of other ECM macromolecules, such as heparin sulfateproteoglycans, which are known to specifically interact with collagen toform ECM.

About 80% inhibition of glycine incorporation was seen for both CDP andNCDP proteins in the presence of 50 ng/ml Halofuginone. The inhibitoryeffect of Halofuginone on deposition of collagenase-digestible ECMproteins was more pronounced with glycine than with sulfate labeledmatrix since unlike glycine, sulfate is incorporated primarily intoglucosaminoglycans which are not degraded by collagenase. A profoundinhibition of ECM deposition was supported by a microscopic examinationof the denuded culture dishes, revealing a thin or non-existant layer ofECM produced in the presence of Halofuginone.

EXAMPLE 6 Halofuginone as a Pretreatment

As noted above, Halofuginone has unexpectedly been shown to be effectiveas a pretreatment for the prevention of a surgically-inducedpathological process, the formation of adhesions. Such an effect alsohas implications for the prevention of surgically-induced keloids andhypertrophic scars. The results of pretreatment with Halofuginone aregiven in Table 1 below.

The experimental method was as follows. Halofuginone was administered inthe diet of two groups of rats at a concentration of 5 mg/kg dry feedfor 4 days before surgery, as a pretreatment. Two other groups of ratswere fed a normal diet and served as control groups. One of the groupsof rats fed Halofuginone and one control group then underwent surgery,which was performed as follows. First, the abdomen of the rats wasshaved and prepared with iodine and alcohol. The abdominal cavity wasentered through a mid-line incision. The small intestine was scrapedfrom the duodenum down to from about 9 to about 10 cm from the cecum,until capillary bleeding occurred. To avoid drying, Hartman's solutionat about 37 C. was occasionally dripped on the intestine. Afterreplacement of the intestine into the abdominal cavity, the abdomen wasclosed in two layers with continuous 00 chromic catgut suture. Thismethod has been previously demonstrated to cause abdominal adhesions(Rivkind, A. I. et al., Eur. Surg. Res., Vol 17, p. 254-258, 1985].

In those rats receiving the drug, Halofuginone treatment was continuedfor 21 days following surgery. At the end of 21 days, the rats wereweighed and the number and severity of adhesions were determinedaccording to a double-blind procedure, in which adhesions wereclassified according to the following grading: 0=no adhesions; I=a thin,filmy, easily separated adhesion; II=several thin adhesions; III=athick, broad adhesion and IV=several thick adhesions. Clearly, 0 is theleast severe and IV is the most severe grade.

Table 1 shows the effect of Halofuginone on adhesion formation. None ofthe rats without surgical intervention, either with or withoutHalofuginone treatment, had any adhesions. However, with one exception,all of the rats which underwent surgical intervention, but which werenot treated with Halofuginone, had at least one adhesion. Most of theadhesions were between loops of the small bowels and at least one wasbetween the small bowel and the omentum. By contrast, a smaller numberHalofuginone-treated rats which underwent surgery had an adhesion, andthe adhesions were more mild. Thus, clearly Halofuginone administered inthe diet, similar to the administration to chickens as a coccidostat,was able to inhibit post-surgical adhesion formation in rats.Furthermore, weight gain by all of the different groups of rats wassubstantially similar, showing that the effect of Halofuginone wasspecific and did not result in any general reduction in overallwell-being of the rats.

TABLE 1 Effect of Halofuginone in Diet on Adhesion Number and SeverityGroup Weight Gain Adhesion Number (g/21 days) Score 1 70 1-2 1 73 1 1 800 1 87 1-2 1 93 3-4 1 83 3 1 92 1-2 1 109 1-2 1 113 3 1 77 1-2 2 86 0-12 76 0 2 61 1 2 80 1-2 2 80 0 2 65 0 2 90 1 2 80 0-1 2 79 0-1 2 63 1-2 3102 0 3 70 0 3 73 0 3 98 0 3 72 0 3 90 0 3 80 0 3 76 0 3 85 0 3 83 0 493 0 4 80 0 4 85 0 4 115 0 4 100 0 4 60 0 4 100 0 4 102 0 4 105 0 4 68 0

EXAMPLE 7 Suitable Formulations for Administration of Halofuginone

Halofuginone can be administered to a subject in a number of ways, whichare well known in the art. Hereinafter, the term “subject” refers to thehuman or lower animal to whom Halofuginone was administered. Forexample, administration may be done topically (including ophtalmically,vaginally, rectally, intranasally), orally, or parenterally, for exampleby intravenous drip or intraperitoneal, subcutaneous, or intramuscularinjection.

Formulations for topical administration may include but are not limitedto lotions, ointments, gels, creams, suppositories, drops, liquids,sprays and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets,capsules or tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable.

Formulations for parenteral administration may include but are notlimited to sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Dosing is dependent on the severity of the symptoms and on theresponsiveness of the subject to Halofuginone. Persons of ordinary skillin the art can easily determine optimum dosages, dosing methodologiesand repetition rates.

EXAMPLE 8 Methods of Treatment of Skin Disorders

As noted above, Halofuginone has been shown to be an effective inhibitorof the clinical etiology of skin disorders, such as keloid andhypertrophic scar formation. The following examples are illustrationsonly of methods of treating skin disorders characterized by abnormalskin cell behavior with Halofuginone, and are not intended to belimiting.

The method includes the step of administering Halofuginone, in apharmaceutically acceptable carrier as described in Example 7 above, toa subject to be treated. Halofuginone is administered according to aneffective dosing methodology, preferably until a predefined endpoint isreached, such as the absence of symptoms of a skin disorder in thesubject. For example, if a subject already had a keloid, the endpointcould be the reduction in size of the keloid or its elimination.

Halofuginone can also be used as a pretreatment, administered to asubject before surgery to substantially prevent the formation of keloidsor hypertrophic scars. Of course, such a pretreatment would be mosteffective for scheduled surgery, as that would allow Halofuginone to beadministered for a sufficient period of time before surgery to be mosteffective.

EXAMPLE 9 Method of Manufacture of a Medicament Containing Halofuginone

The following is an example of a method of manufacturing Halofuginone.First, Halofuginone is synthesized in accordance with goodpharmaceutical manufacturing practice. Examples of methods ofsynthesizing Halofuginone, and related quinazolinone derivatives, aregiven in U.S. Pat. No. 3,338,909. Next Halofuginone is placed in asuitable pharmaceutical carrier, as described in Example 7 above, againin accordance with good pharmaceutical manufacturing practice.

While the invention has been described with respect to a limited numberof embodiments, it will be appreciated that many variations,modifications and other applications of the invention may be made.

1. A method for the treatment of psoriasis in a subject, the methodcomprising the step of administering to the subject a pharmaceuticallyeffective amount of a compound having a formula:

wherein: n is 1 or 2; R₁ is a member of the group consisting ofhydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy, and R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl.
 2. The method of claim 1, wherein said compound isHalofuginone.
 3. The method of claim 1, wherein said compound isadministered to the subject by a route selected from the groupconsisting of oral, parenteral and topical.
 4. The method of claim 3,wherein said compound is administered to the subject by topicalapplication.
 5. The method of claim 4, wherein said compound iscontained in a pharmaceutical carrier selected from the group consistingof a lotion, an ointment, a gel, a cream, a liquid and a spray.
 6. Themethod of claim 5, wherein the step of administering said compound tothe subject is performed until symptoms of psoriasis are absent in thesubject.